Aggregate-free urate oxidase for preparation of non-immunogenic polymer conjugates

• US 6,783,965 B1
• Filed: 02/10/2000
• Issued: 08/31/2004
• Est. Priority Date: 02/10/2000
• Status: Expired due to Term

• Current Assignee: Horizon Therapeutics USA, Inc. (Amgen Inc.), Duke University
• Original Assignee: Mountain View Pharmaceuticals, Inc.
• Inventors: Sherman, Merry R., Saifer, Mark G. P., Williams, L. David
• Primary Examiner(s): Achutamurthy, Ponnathapu
• Assistant Examiner(s): PAK, YONG D
• Application Number:
  US09/501,730
• Time in Patent Office: 1,664 Days
• Field of Search: 435/190, 435/191, 435/440, 435/170, 424/94.4, 424/94.6, 536/23.2, 530/350, 530/413
• US Class Current: 435/190
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 47/60 the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
• CPC Class Codes: A61P 13/00 Drugs for disorders of the urinary system
• CPC Class Codes: A61P 13/12 of the kidneys
• CPC Class Codes: A61P 19/06 Antigout agents, e.g. antihyperuricemic or uricosuric agents
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C12N 9/0046 with oxygen as acceptor (1.7.3)
• CPC Class Codes: C12N 9/96 Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates

Abstract

A naturally occurring or recombinant protein, especially a mutein of porcine urate oxidase (uricase), that is essentially free of large aggregates can be rendered substantially non-immunogenic by conjugation with a sufficiently small number of strands of polymer such that the bioactivity of the protein is essentially retained in the conjugate. Such conjugates are unusually well suited for treatment of chronic conditions because they are less likely to induce the formation of antibodies and/or accelerated clearance than are similar conjugates prepared from protein preparations containing traces of large aggregates.

Claims

1. Purified urate oxidase (uricase) that contains no more than about 2% of aggregates larger than octamers, wherein greater than about 20% of said uricase is in the tetrameric or octameric form.

2. The uricase of claim 1, wherein the uricase is mammalian uricase.

3. The uricase of claim 2, wherein the uricase is porcine liver, bovine liver or ovine liver uricase.

4. The uricase of claim 1, wherein the uricase is recombinant.

5. The uricase of claim 4, wherein the uricase has the sequence of porcine, bovine, ovine or baboon liver uricase.

6. The uricase of claim 4, wherein the uricase is chimeric.

7. The uricase of claim 6, wherein the chimeric uricase contains portions of porcine liver and baboon liver uricase.

8. The uricase of claim 7, wherein the chimeric uricase is porcine uricase in which arginine residue 291 of SEQ ID NO:

2 has been replaced by lysine (R291 K) and threonine residue 301 of SEQ ID NO;

2 has been replaced by serine (T301 S) (PKS uricase).

9. The uricase of claim 4, wherein the uricase has the sequence as set forth in SEQ ID NO:

2, wherein tyrosine 97 has been replaced by histidine.

10. The uricase of claim 1, wherein the uricase is a fungal or microbial uricase.

11. The uricase of claim 10, wherein the fungal or microbial uricase is isolated from Aspergillus flavus, Arthrobacter globiformis, Bacillus sp. or Candida utilis, or is a recombinant enzyme having the sequence of one of said uricases.

12. The uricase of claim 1, wherein the uricase is an invertebrate uricase.

13. The uricase of claim 12, wherein the invertebrate uricase is isolated from Drosophila melanogaster or Drosophila pseudoobscura, or is a recombinant enzyme having the sequence of one of said uricases.

14. The uricase of claim 1, wherein the uricase is a plant uricase.

15. The uricase of claim 14, wherein the plant uricase is isolated from root nodules of Glycine max or is a recombinant enzyme having the sequence of said uricase.

16. A uricase conjugate comprising the uricase of claim 1 conjugated to poly(ethylene glycol) or poly(ethylene oxide).

17. The uricase conjugate of claim 16, wherein said poly(ethylene glycol) is monomethoxy poly(ethylene glycol).

18. The uricase conjugate of claim 16, wherein said uricase is conjugated to said poly(ethylene glycol) or poly(ethylene oxide) via a linkage selected from the group consisting of urethane (carbamate), secondary amine and amide.

19. The uricase conjugate of claim 16, wherein said poly(ethylene glycol) or poly(ethylene oxide) has a molecular weight between about 5 kDa and 30 kDa.

20. The uricase conjugate of claim 19, wherein said poly(ethylene glycol) or poly(ethylene oxide) has a molecular weight between about 10 kDa and 20 kDa.

21. The uricase conjugate of claim 16, wherein the average number of strands of said poly(ethylene glycol) or poly(ethylene oxide) is between about 2 and 12 per uricase subunit.

22. The uricase conjugate of claim 21, wherein the average number of strands of said poly(ethylene glycol) or poly(ethylene oxide) is between about 6 and 10 per uricase subunit.

23. The uricase conjugate of claim 22, wherein the average number of strands of said poly(ethylene glycol) or poly(ethylene oxide) is between about 7 and 9 per uricase subunit.

24. The uricase conjugate of claim 16, wherein the poly(ethylene glycol) or poly(ethylene oxide) is linear.

25. The uricase conjugate of claim 16, wherein the poly(ethylene glycol) or poly(ethylene oxide) is branched.

26. A pharmaceutical composition for lowering uric acid levels in a body fluid or tissue, comprising the conjugate of claim 16 and a pharmaceutically acceptable carrier.

27. The pharmaceutical composition of claim 26, wherein said composition is stabilized by lyophilization and dissolves upon reconstitution to provide solutions suitable for parenteral administration.

29. The purified uricase of claim 1, wherein about 98% to about 100% of said uricase is in the tetrameric or octameric form.

28. A purified fragment of uricase that contains no more than about 2% of aggregates larger than octamers, wherein said fragment is a recombinant uricase that has been truncated at the amino terminus, at the carboxyl terminus, or at both the amino and carboxyl termini, and wherein greater than about 20% of said truncated uricase is in the tetrameric or octameric form.

30. Isolated uricase prepared by a method comprising separating uricase aggregates larger than octamers from uricase tetramers and octamers and excluding such aggregates from the isolated uricase, wherein about 98% to about 100% of said uricase is in the tetrameric or octameric form.